Attenuated Recombinant Alphaviruses Incapable of Replicating in Mosquitoes and Uses Thereof

ABSTRACT

The present invention discloses an attenuated recombinant alphavirus that is incapable of replicating in mosquito cells and of transmission by mosquito vectors. These attenuated alphavirus may include but is not limited to Western Equine Encephalitis virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus or Chikungunya virus. The present invention also discloses the method of generating such alphaviruses and their use as immunogenic compositions.

CROSS-REFERENCE TO RELATED APPLICATION

This continuation-in-part application claims benefit of priority underU.S.C. §120 of international application PCT/US2009/000458, filed Jan.23, 2009, which claims benefit of priority of provisional applicationU.S. Ser. No. 61/062,229 filed on Jan. 24, 2008, now abandoned, theentirety of which is incorporated by reference.

FEDERAL FUNDING LEGEND

This invention was produced in part using funds obtained through a award1U54 AI057156 from the National Institute of Health/National Instituteof Allergy and Infectious Disease. Consequently, the federal governmenthas certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the fields of molecular biology,virology and immunology of alphaviruses. More specifically, the presentinvention provides an attenuated, recombinant alphaviruses havingmosquito infection incompetence and discloses a method of generatingsuch alphaviruses and use of these attenuated alphaviruses inimmunogenic compositions.

2. Description of the Related Art

The Alphavirus genus in the Togaviridae family contains a number ofsignificant human and animal pathogens. These viruses are widelydistributed on all continents except for the Antarctic region, andrepresent a significant public health threat (18, 39). Under naturalconditions, most of the alphaviruses are transmitted by mosquitoes, inwhich they cause a persistent, life-long infection that has littleeffect on the biological functions of the vector. In vertebratesinfected by mosquitoes during their blood meal, alphaviruses cause anacute infection, characterized by a viremia that is a prerequisite ofinfection of new mosquitoes and its circulation in nature.

Venezuelan equine encephalitis virus (VEEV) is one of the mostpathogenic members of the alphavirus genus. It continuously circulatesin South, Central and North America and causes sporadic epidemics andepizootics that involve humans, horses and other domestic animals.During the most recent major outbreak in Venezuela and Colombia (1995)involving subtype IC VEEV, about 100,000 human cases occurred, with over300 fatal encephalitis cases estimated (37). During VEEV epizootics,equine mortality due to encephalitis can reach 83%, and while theoverall mortality rate is low in humans (<1%), neurological disease,including disorientation, ataxia, mental depression, and convulsions,can be detected in up to 14% of infected individuals, especiallychildren (21). The human disease caused by VEEV is characterized as afebrile illness with chills, severe headache, myalgia, somnolence andpharyngitis. Young and old individuals develop a reticuloendothelialinfection with severe lymphoid depletion, followed by encephalitis. Theresult of the CNS infection is an acute meningoencephalitis that leadsto the death of neuronal cells (9). The neurologic signs appear within4-10 days of the onset of illness and include seizures, paresis,behavioral changes and coma.

In spite of the continuous threat of VEEV epidemics, no safe andefficient vaccines have been designed for this virus. The attenuatedTC-83 strain of VEEV was developed more than four decades ago by serialpassage of a highly virulent Trinidad donkey (TRD) strain of VEEV inguinea pig heart cells (4). Presently, TC-83 is still the only availablevaccine for laboratory workers and military personnel. Over 8,000 peoplehave been vaccinated (2, 8, 34), and the cumulative data unambiguouslydemonstrate that nearly 40% of all vaccinees develop a disease with somesymptoms typical of natural VEE, including fever, systemic illness andother adverse effects (2). This TC-83 strain universally kills newborn,but not adult, mice after i.c. and s.c. inoculation (31), and is thus agood starting material for further attenuation and study of the effectsof the mutations on viral pathogenesis.

The VEEV genome is a nearly 12-kb-long, single-stranded RNA molecule ofpositive polarity that mimics the structure of cellular mRNAs. Thegenome RNA contains both a 5′ methylguanylate cap and a 3′ polyadenylatetail (24), features which allow translation of viral proteins by hostcell machinery immediately after release of the genome RNAs from thenucleocapsids. The 5′ two-thirds of the genome is translated into thenonstructural proteins (nsPs) that comprise the viral components of thereplicative enzyme complex required for replication of the viral genomeand transcription of the subgenomic RNA. The subgenomic RNA correspondsto the 3′ third of the genome. It is synthesized from the subgenomicpromoter and translated into the viral structural proteins. Theattenuated phenotype of the VEEV strain TC-83 is the result of twomutations in the strain TRD genome: one of them replaced an amino acidat position 120 in E2 glycoprotein, and the second changed nt 3 in the5′UTR (11, 23, 24, 43). Thus, because of the alphavirus' very highmutation rate, the reversion of TC-83 to a pathogenic phenotype remainsa great concern in the event that the appropriate selective conditions,such as virus passage in vivo, would occur. Moreover, VEEV TC-83 iscapable of replicating in mosquito cells, and infecting mosquitoesfollowing vaccination (32); therefore, its transmission by mosquitoesremain possible.

Ideally, live arbovirus vaccine strains should not be transmissible byarthropod vectors, because circulation among reservoir hosts could leadto unforeseen changes that might include increased virulence. This isespecially true for attenuated strains, produced from wild-type virusesthat rely on small numbers of attenuating mutations that may be subjectto reversion, or for genetically modified strains that might evolve inunanticipated ways if they underwent vector-borne circulation. Theformer risk was underscored by the detection of the VEEV TC-83 vaccinestrain in mosquitoes collected in Louisiana during 1971 (32), an areaoutside the epizootic/epidemic that was restricted to Texas.

The development of infectious cDNA for alphaviruses opened anopportunity to explore their attenuation by extensively modifying theviral genomes, an approach that might minimize or exclude the reversionto the wt, pathogenic phenotype. Moreover, the genomes of suchalphaviruses can be engineered to contain RNA elements that would befunctional only in cells of vertebrate, but not insect, origin. Thus,such extensive mutations could prevent transmission of the geneticallymodified viruses by mosquito vectors.

Despite its importance as an emerging human and animal pathogen, itspotential as a biological weapon and concerns about application ofattenuated alphaviruses, the prior art is deficient in methods ofgenerating attenuated strains of alphaviruses that are capable ofreplicating only in vertebrate cells. The present invention fulfillsthis long-standing need and desire in the art.

SUMMARY OF THE INVENTION

In one embodiment of the present invention, there is provided a methodof generating an attenuated recombinant alphavirus. Such a methodcomprises cloning the internal ribosomal entry site ofencephalomyelocarditis virus (EMCV IRES) between the end ofnon-structural protein 4 (nsP4) coding sequence and initiating AUG of asubgenomic RNA coding sequence of an alphavirus instead of natural 5′UTR of subgenomic RNA. The subgenomic promoter is inactivated with alarge number of synonymous mutations such that the IRES must be retainedto drive expression of the structural proteins. In a further relatedembodiment of the present invention, there is an attenuated recombinantalphavirus generated by the method discussed supra.

In another embodiment of the present invention, is provided a method ofgenerating attenuated, recombinant alphavirus, comprising the steps ofretaining the subgenomic promoter and positioning the capsid proteingene downstream of the envelope protein genes; and cloning internalribosomal entry site of encephalomyelocarditis virus (EMCV IRES) betweenthe end of the envelope protein genes and the capsid protein gene.

In another embodiment of the present invention, there is provided amethod of generating attenuated, recombinant alphavirus, comprising thesteps of: cloning an internal ribosomal entry site of aencephalomyelocarditis virus between the end of nonstructural protein 4coding sequence and initiating AUG of a subgenomic RNA coding sequenceof an alphavirus, instead of a natural 5′ UTR of the subgenomic RNA;inactivating the subgenomic promoter of the alphavirus by clusteredpoint mutations and deletion of the natural 5′UTR in the subgenomic RNA;positioning the capsid protein gene downstream of the inactivatedsubgenomic promoter under control of the internal ribosomal entry siteof said encephalomyelocarditis virus; and positioning the envelopeglycoprotein genes under a subgenomic promoter cloned downstream of thetermination codon of the capsid gene, thereby generating the attenuated,recombinant alphavirus.

In yet another related embodiment of the present invention, there isprovided a vector comprising a nucleotide sequence encoding theattenuated recombinant alphavirus and a host cell comprising andexpressing this vector. In a yet another related embodiment of thepresent invention, there is provided a pharmaceutical composition. Thiscomposition comprises the attenuated recombinant alphavirus discussedsupra and a pharmaceutically acceptable carrier. In a related embodimentof the present invention, there is provided an immunogenic composition.This immunogenic composition comprises the attenuated recombinantalphavirus described herein.

In another related embodiment of the present invention, there isprovided a method of protecting a subject from infections resulting fromexposure to an alphavirus. Such a method comprises administering animmunologically effective amount of the immunogenic compositioncomprising the attenuated, recombinant alphavirus described herein,thereby protecting the individual from the infections resulting from theexposure to the alphavirus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show replication of the recombinant, EMCV IRES-encoding,VEEV TC-83-derived viruses in BHK-21 cells. FIG. 1A is a schematicrepresentation of the designed viral genomes, infectivities of the invitro-synthesized RNAs in the infectious center assay, virus titers at24 h post transfection of 1 mg of the in vitro-synthesized RNAs intoBHK-21 cells, and sizes of the plaques, formed by indicated viruses inBHK-21 cells at 48 h post transfection. Arrows indicate functionalsubgenomic promoters. Filled boxes indicate EMCV IRES. FIG. 1B showsalignment of the subgenomic promoter-containing fragment (SEQ D NO: 1)of the VEEV TC-83 genome and the corresponding fragment of theVEEV/mutSG/IRES (SEQ ID NO: 2). The position of the promoter isindicated by open box. The start of the subgenomic RNA in the VEEV TC-83genome and the beginning of the EMCV IRES are indicated by arrows. Themutations, introduced into the VEEV/mutSG/IRES genome are shown in lowercase letters. FIG. 1C shows plaques, formed in BHK-21 cells by viruses,harvested at 24 h post transfection. FIG. 1D shows replication of theviruses after transfection of 1 mg of the in vitro-synthesized RNAs intoBHK-21 cells.

FIGS. 2A-2C show mutations found in the plaque-purified VEEV/mutSG/IRESvariants, which demonstrated more efficient replication in BHK-21 cells,and the effect of the defined adaptive mutations on VEEV TC-83 andVEEV/mutSG/IRES replication. FIG. 2A shows the list of the mutations,found in the genomes of plaque isolates, compared to the publishedsequence of VEEV TC-83 (24). FIG. 2B is a schematic representation ofthe VEEV TC-83 and VEEV/mutSG/IRES genomes, having either one or bothidentified mutations, and the infectivity of the in vitro-synthesizedviral RNAs in the infectious center assay. Functional subgenomicpromoters are indicated by arrows, and EMCV IRES by filled boxes. FIG.2C shows replication of the designed viruses in BHK-21 cells aftertransfection of 1 mg of the in vitro-synthesized viral genomes.

FIGS. 3A-3B shows mutations identified in the nsP2 protein ofVEEV/mutSG/IRES variants demonstrating a large-plaque phenotype. FIG. 3Ashows a list of the mutations identified in the genomes of theplaque-purified isolates from virus stock, harvested at 24 h posttransfection of the in vitro-synthesized RNA (Orig.), and in the genomesof isolates from the stock that was additionally passaged three times inVero cells (Pass.). FIG. 3B shows localization of the defined mutationsin the VEEV nsP2. The positions of currently known functional domains inalphavirus nsP2 (38, 39) are indicated.

FIGS. 4A-4B show an analysis of protein and RNA synthesis in BHK-21cells transfected with the in vitro-synthesized recombinant viral RNAs.Cells were electroporated with 4 mg of the indicated RNAs and seededinto 35-mm dishes. In FIG. 4A, at 4.5 h post transfection, medium in thewells was replaced by 1 ml of aMEM supplemented with 10% FBS, ActD (1mg/ml) and [³H]uridine (20 mCi/ml). After 4 h of incubation at 37° C.,RNAs were isolated and analyzed by agarose gel electrophoresis. Thepositions of viral genomic and subgenomic RNAs are indicated by G andSG, respectively. The VEEV/IRES-specific subgenomic RNA forms a morediffuse band than do other, subgenomic RNA-producing, viruses, because,in the gel, it co-migrates with the ribosomal 28S RNA. In FIG. 4B, at 12h post transfection, protein were metabolically labeled with[³⁵S]methionine and analyzed on a sodium dodecyl sulfate-10%polyacrylamide gel. The positions of molecular weight markers (kDa) areindicated at the left side of the gel. The positions of viral structuralproteins: C, E1 and p62 (the precursor of E2) are shown at the rightside of the gel. Asterisks indicate the positions of cellular proteins(the heat-shock proteins), induced by replication of the IRES-encodingviruses.

FIGS. 5A-5C show passaging of the recombinant, EMCV IRES-containing VEEVvariants in C₇10 cells. FIG. 5A is a schematic representation of viralgenomes. Arrow indicates the position of the functional subgenomicpromoter. The filled box indicates the position of EMCV IRES. FIG. 5Bshows titers of the recombinant viruses after passaging in C₇10 cells.Cells in 35-mm dishes were infected with 400 ml of virus samplesharvested either at 24 h post transfection of BHK-21 cells with the invitro-synthesized RNA (P1) or 48 h post infection of C₇10 cells. Dashedline indicates the limit of detection. FIG. 5C shows the deletions ofthe IRES-containing sequence (SEQ ID NOS: 4-5) identified in theplaque-purified VEEV/IRES variants, demonstrating efficient replicationin C₇10 cells. The residual EMCV IRES-specific sequences are indicatedby lower case letters. The VEEV/IRES variants are aligned with TC-83strain of VEEV (SEQ ID NO: 6).

FIG. 6 shows replication of VEEV/mutSG/IRES/1 and VEEV TC-83 in the NIH3T3 cells. Cells were infected at an MOI of 10 PFU/cell. Media werereplaced at the indicated time points, and virus titers were measured byplaque assay on BHK-21 cells. The same samples were used to measureIFN-a/b release in biological assay. Concentrations of released IFN-a/bare presented in international units (IU) per ml.

FIG. 7 shows survival of mice infected with VEEV TC-83 andVEEV/mutSG/IRES/1 viruses. Six-day-old NIH Swiss mice were inoculatedi.c. with ca. 10⁶ PFU of the indicated viruses. Animals were monitoredfor two months. No deaths occurred after day 9 post-infection in any ofthese experiments.

FIG. 8 shows survival following vaccination and challenge of adult mice.Five-to-6-week-old female NIH Swiss mice were immunized s.c. with VEEVstrain TC-83 or the recombinant virus at a dose of ca. 10⁶ PFU. Threeweeks after immunization, mice were challenged s.c. with ca. 10⁴ PFU ofVEEV strain 3908, and mortality was recorded.

FIG. 9 shows a schematic method of generating attenuated, recombinantalphavirus incapable of infecting mosquitoes.

FIG. 10 is a schematic representation showing genetic strategy forchikungunya virus (CHIKV) CHIK/IRES constructs.

FIG. 11 shows Viremia titers in 6-day-old CD1 mice after subcutaneousinfection with CHIKV strains (10⁶ PFU). Dashed line is the limit ofdetection in the plaque assays.

FIG. 12 shows Virus titers in the legs of 6-day-old CD1 mice aftersubcutaneous infection with CHIKV strains (10⁶ PFU).

FIG. 13 shows virus titers in the brains of 6-day-old CD1 mice aftersubcutaneous infection with CHIKV strains (10⁶ PFU). Dashed line is thelimit of detection in the plaque assays.

FIG. 14 depicts survival of adult CD1 mice 4 weeks after vaccinationwith 105 PFU and intranasal challenge with the Ross strain of CHIKV.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is drawn to a new strategy of developingattenuated strains of alphaviruses. These attenuated alphaviruses arecapable of replicating efficiently only in vertebrate cells. Thisphenotype was achieved by rendering the translation of the viralstructural proteins and ultimately, viral replication dependent on theinternal ribosome entry site of encephalomyocarditis virus (EMCV IRES).Such a recombinant virus is viable and demonstrated highly attenuatedphenotype in newborn mice, yet induces protective immunity against VEEVinfection and CHIKV. Further, formalin-inactivated vaccines areexpensive and inefficient. In addition to this, these vaccines alsorequire multiple, repeated vaccinations. Furthermore, the only availablelive attenuated vaccine against VEEV infection is very reactogenic andinfects mosquitoes during the blood meal on vaccinated horses. Theattenuated alphaviruses discussed herein provide a significant advantageover the vaccines that are currently available since the attenuatedphenotype is irreversible. Further, the genetically engineeredalphaviruses are not able to replicate in mosquito cells, thus,suggesting a new approach for generating new, live recombinant viruses,which are not capable of replicating in mosquitoes and thus, incapableof circulating in nature.

The development of the infectious cDNA clones of Sindbis and otheralphaviruses (12, 25, 36) opened the opportunity not only for thereverse genetics experiments aimed at studying different aspects of thevirus biology and pathogenesis, but also for the development of newrecombinant vaccines. Attenuation of the viruses by passaging either intissue culture or in chicken embryos (29) generally results fromaccumulation of small numbers of point mutations in the structural andnonstructural genes, and in the cis-acting elements of viral genomes.For example, the VEEV TC-83 vaccine strain relies on only 2 pointmutations for its attenuation, and the high degree of reactogenicity(34) probably reflects the instability of this attenuation mechanism.This raises a concern about the possible reversion to the wt, pathogenicphenotype during virus replication in vaccinated individuals. The numberof mutations can be additionally increased by chemical mutagenesis (28),but this procedure also does not make the introduced changesirreversible. The genetic manipulations with infectious cDNA clones ofthe RNA⁺ viruses open great possibilities for stronger modification ofviral genomes, and provide an opportunity to introduce, extensivedeletions that would make it impossible to revert to the wt genomesequence (5, 6, 10, 19), or additional genetic material that mightenhance the immunogenicity of the variants.

There is also a great concern that genetically altered arboviruses mightbe introduced into the natural circulation, mediated by mosquitovectors, and demonstrate a further evolution during long-termreplication, either in mosquitoes or during viremia development invertebrate hosts. An example is the use of VEEV TC-83, which is capableof producing levels of viremia in equids sufficient for infectingmosquitoes. The isolation of TC-83 from naturally infected mosquitoescollected in Louisiana (32) during the 1971 Texas epidemic underscoredthe risk of transmission of the attenuated alphaviruses. Therefore, indesigning a new generation of live vaccine strains, it is prudent tomake arboviruses not only highly attenuated, but also capable ofreplicating only in cells of vertebrate origin. This can be achieved bycloning cell-specific RNA elements into viral genomes. In contrast tothe cricket paralysis virus IRES (20), the EMCV-specific element wasexpected to function very inefficiently in arthropod cells.

In the present invention, the EMCV IRES was cloned into VEEV TC-83genome and the chikungunya virus genome to make the translation of viralstructural genes IRES-dependent. One of the genomes contained afunctional subgenomic promoter, and the IRES in the 5′UTR of thesubgenomic RNA. This virus was viable, but its ability to produce thesubgenomic RNA promoted further evolution, which resulted in IRESdeletion and reversion, most likely, to a standard, cap-dependenttranslation of the structural proteins. The latter deletions made itcapable of replicating in mosquito cells. The second variant withmultiple mutations to inactivate the subgenomic promoter was stable interms of its inability to revert to a cap-dependent translation. Becausesuch reversion would not only require the IRES deletion, but also therestoration of the subgenomic promoter, which was inactivated by 13mutations, direct reversion of these multiple mutations probablyrepresents a negligible risk. However, this variant of TC-83 developedan interesting way to evolve to a more efficiently replicating phenotypeby accumulating additional, adaptive mutations in the nsP2 gene. Thesemutations did not noticeably change the level of viral RNA replication,synthesis of the viral structural proteins, or theircompartmentalization in the cells. The detected mutations also did notcreate an additional signal that could increase the efficiency of thegenome packaging. Thus, the mechanism of their functioning remains to bedetermined. However, the accumulating published data suggest that thepackaging of the genomes of the RNA⁺ viruses is strongly determined bythe replicative complexes, and the genomes need to be presented by thefunctional nsPs to the structural proteins for particle formation (22,30). The working hypothesis herein is that the helicase domain of thensP2 might be involved in the viral genome's presentation for itspackaging into the nucleocapsids, and, thus, the identified mutationscould have a positive effect on the efficiency of this process.

The present invention sought to develop VEEV and CHIKV variants that areincapable of replicating in arthropod vectors and demonstrate a stable,more attenuated phenotype. Slower growth of the designedVEEV/mutSG/IRES/1 variant in both IFN-alpha/beta-competent and IFNsignaling-deficient BHK-21 cells, its ability to induce higher levels ofIFN-alpha/beta in tissue culture, its greatly reduced ability to killnewborn mice even after i.c. inoculation, and its inability to replicatein mosquito cells suggest that this variant might meet thoserequirements. Its immunogenicity will be further investigated indifferent animal models. Moreover, it is believed that otherencephalogenic alphaviruses can be attenuated by using a similar, EMCVIRES-based strategy, which can be applied in combination with otherapproaches that have been developed within the recent years (1, 13-15,31).

In summary, the present invention describes the development ofattenuated alphaviruses and their application as a new type of vaccineagainst the encephalitogenic alphaviruses that include VEEV, EEEV andWEEV and other alphaviruses such as Chikungunya virus that cause diseasein humans and livestock. Replication of such alphaviruses would dependon EMCV IRES, that makes them incapable of replicating in mosquito cellsor mosquito vectors. More importantly, this phenotype is irreversiblebecause of the extensive modifications introduced into viral genome.Therefore, these new variants can be used for vaccination without aconcern about possibility of their transmission by natural mosquitovectors.

The present invention is directed to generating attenuated, recombinantalphavirus, comprising the step of: cloning the internal ribosomal entrysite of encephalomyelocarditis virus (EMCV IRES) between the end ofnonstructural protein (nsP4) coding sequence and initiating AUG of asubgenomic RNA coding sequence of an alphavirus instead of natural 5′UTR. This method may further comprise inactivating the subgenomicpromoter of the alphavirus by clustered point mutations and deletion ofthe natural 5′ UTR in the subgenomic RNA. Further, the inactivation ofthe subgenomic promoter may not modify the carboxy terminus ofnon-structural protein 4. Additionally, the method may further compriseintroducing adaptive mutations in non-structural protein 2 (nsP2)effective to increase virus replication, release and virus titers.Examples of the adaptive mutations in non-structural protein 2 mayinclude but are not limited to to Y₃₇₀→C, K₃₇₁→Q, P₃₄₉→T, D₄₁8→A, K₄₂₃→Tor combinations thereof. Furthermore, examples of the alphavirus mayinclude but is not limited to Venezuelan Equine Encephalitis virus(VEEV), Eastern Equine Encephalitis Virus (EEEV), Western EquineEncephalitis virus (WEEV) or Chikungunya virus.

In an embodiment of the present invention, there is provided a method ofgenerating attenuated, recombinant alphavirus, comprising the steps ofpositioning the capsid protein gene downstream of the envelope proteingenes; and cloning internal ribosomal entry site ofencephalomyelocarditis virus (EMCV IRES) between the end of the envelopeprotein genes and the capsid protein gene.

In another embodiment of the present invention, there is provided amethod of generating attenuated, recombinant alphavirus, comprising thesteps of: cloning an internal ribosomal entry site of aencephalomyelocarditis virus between the end of nonstructural protein 4coding sequence and initiating AUG of a subgenomic RNA coding sequenceof an alphavirus, instead of a natural 5′ UTR of the subgenomic RNA;inactivating the subgenomic promoter of the alphavirus by clusteredpoint mutations and deletion of the natural 5′UTR in the subgenomic RNA;positioning the capsid protein gene downstream of the inactivatedsubgenomic promoter under control of internal ribosomal entry site ofsaid encephalomyelocarditis virus; and positioning envelope glycoproteingenes under a subgenomic promoter cloned downstream of the terminationcodon of capsid gene, thereby generating the attenuated, recombinantalphavirus.

The present invention is also directed to an attenuated recombinantalphavirus generated by one of the methods described herein. Such analphavirus may be incapable of replicating in mosquitoes, incapable oftransmission by mosquito vectors, capable of inducing high levels of IFNalpha/beta, slow growth in IFN alpha/beta cells, slow growth in IFNsignaling-deficient BHK-21 cells or a combination thereof.

The present invention is still further directed to a vector comprising anucleotide sequence encoding the attenuated recombinant alphavirusgenerated by one of the methods described hereinand a host cellcomprising and expressing the vector. Constructing vectors andexpressing them in cells is well-known and standardized technique in theart. Hence, one of skill in the art may construct such vectors based onroutine experimentation and knowledge that is available in the art.

The present invention is further directed to a pharmaceuticalcomposition, comprising the attenuated recombinant alphavirus discussedsupra and a pharmaceutically acceptable carrier. The present inventionis also directed to an immunogenic composition, comprising an attenuatedrecombinant alphavirus generated by one of the methods described herein.

The present invention is still further directed to a method ofprotecting a subject from infections resulting from exposure to analphavirus, comprising the step of: administering immunologicallyeffective amount of the immunogenic composition discussed supra, therebyprotecting the subject from infections resulting from exposure to thealphavirus. The subject benefiting from such a method may be human orlivestock.

The present invention is still further directed to a method ofgenerating attenuated, recombinant alphavirus incapable of infectingmosquitoes, comprising the step of cloning the internal ribosomal entrysite of encephalomyelocarditis virus and capsid genes downstream of theenvelope glycoprotein genes with said capsid gene at the 3′ end of thesubgenomic region just upstream of the 3′ UTR, wherein the capsid isexpressed in a cap-independent manner and the envelope protein genes istranslated in a cap-dependent manner but the capsid protein translatedin an IRES-dependent manner.

As used herein, the term, “a” or “an” may mean one or more. As usedherein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Asused herein “another” or “other” may mean at least a second or more ofthe same or different claim element or components thereof.

The composition described herein can be administered independently,either systemically or locally, by any method standard in the art, forexample, subcutaneously, intravenously, parenterally, intraperitoneally,intradermally, intramuscularly, topically, or nasally. Dosageformulations of the composition described herein may compriseconventional non-toxic, physiologically or pharmaceutically acceptablecarriers or vehicles suitable for the method of administration and arewell known to an individual having ordinary skill in this art.

The composition described herein may be administered independently oneor more times to achieve, maintain or improve upon a therapeutic effect.It is well within the skill of an artisan to determine dosage or whethera suitable dosage of the composition comprises a single administereddose or multiple administered doses. An appropriate dosage depends onthe subject's health, the induction of immune response and/or preventionof infection caused by the alphavirus, the route of administration andthe formulation used.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. One skilled in the art will appreciate readilythat the present invention is well adapted to carry out the objects andobtain the ends and advantages mentioned, as well as those objects, endsand advantages inherent herein. Changes therein and other uses which areencompassed within the spirit of the invention as defined by the scopeof the claims will occur to those skilled in the art.

Example 1 Cell cultures

The BHK-21 cells were provided by Paul Olivo (Washington University, St.Louis, Mo.), and the Vero cells by Charles Rice (Rockefeller University,NY, N.Y.). The NIH 3T3 cells were obtained from the American Type TissueCulture Collection (Manassas, Va.). These cell lines were maintained at37° C. in alpha minimum essential medium (aMEM) supplemented with 10%fetal bovine serum (FBS) and vitamins. Mosquito C₇10 cells were obtainedfrom Henry Huang (Washington Univ., St. Louis, Mo.) and propagated inDMEM supplemented with 10% heat-inactivated FBS and 10% tryptosephosphate broth (TPB).

Example 2 Plasmid Constructs

Standard recombinant DNA techniques were used for all plasmidconstructions. The original plasmid with VEEV TC-83 genome under thecontrol of SP6 RNA polymerase promoter, pVEEV TC-83, was described (33).pVEEV/IRES contained EMCV IRES with the first 4 codons of the EMCVpolyprotein. This sequence was cloned into the VEEV subgenomicRNA-coding sequence between the end of the 5′UTR and the initiating AUG.pVEEV/mutSG/IRES encoded the VEEV TC-83 genome, in which the subgenomicpromoter was inactivated by clustered point mutations, which did notmodify the amino acid sequence (SEQ ID NO: 3) of the carboxy terminus ofnsP4 (FIGS. 1A and 1B). This viral genome had the 5′UTR of thesubgenomic RNA deleted. Thus, VEEV TC-83 nonstructural and structuralproteins were expected to be synthesized from the same genomic RNA. Theadaptive mutations were introduced into pVEEV/mutSG/IRES-encoded nsP2 byPCR amplification of the fragments of interest of the selected variants,followed by replacement of the corresponding fragment in the originalgenome. The same PCR-based technique was used for synthesis cloning ofdifferent fragments into the SphI site in the 3′UTR of theVEEV/mutSG/IRES genome. All of the cloned fragments were sequencedbefore further experiments with the rescued viruses.

Example 3 RNA Transcriptions

Plasmids were purified by centrifugation in CsCl gradients andlinearized by Mlul digestion. RNAs were synthesized by SP6 RNApolymerase (Ambion) in the presence of cap analog (New England Biolabs).The yield and integrity of transcripts were analyzed by gelelectrophoresis under non-denaturing conditions. RNA concentration wasmeasured on a Fluor Chem imager (Alpha Innotech), and transcriptionreactions were used for electroporation without additional purification.

Example 4 RNA Transfections

Electroporation of BHK-21 cells was performed under described conditions(27). To rescue the viruses, 1 mg of in vitro-synthesized viral genomeRNA was electroporated into the cells (27), and then they were seededinto 100-mm dishes and incubated until cytopathic effects were observed.Virus titers were determined using a standard plaque assay on BHK-21cells (26).

To assess the RNA infectivity, 10-fold dilutions of electroporatedBHK-21 cells were seeded in 6-well Costar plates containingsubconfluent, naïve cells. After a 1 h incubation at 37° C. in a 5% CO₂incubator, cells were overlaid with 2 ml of MEM-containing 0.5%Ultra-Pure agarose supplemented with 3% FBS. Plaques were stained withcrystal violet after 2 days incubation at 37° C., and infectivity wasdetermined in plaque-forming units (PFU) per mg of transfected RNA.

Example 5 Sequencing of Viral Genomes

Large plaques were randomly selected during titering of viral stocks(without staining with neutral red). Viruses were extracted from theagarose plugs into MEM, and aliquots of the latter media were used toinfect BHK-21 cells in 35-mm dishes. After development of profound CPE,virus stocks were harvested for further characterization, and RNAs wereisolated from the infected cells by TRizol reagent according to theinstructions of the manufacturer (Invitrogen). ˜1000 nt-long,overlapping fragments were synthesized using standard RT-PCR techniques,purified by agarose gel electrophoresis and sequenced. Sequencing of the5′UTR was performed by using a FirstChoice RLM-RACE Kit (Ambion) asdescribed (16).

Example 6 Viral Replication Analysis

One-fifth of the electroporated cells were seeded into 35-mm dishes. Atthe times indicated in the figures, media were replaced and virus titerswere determined by plaque assay on BHK-21 cells (26). Alternatively,BHK-21, NIH3T3 or C₇10 cells were seeded into 35-mm dishes and infectedat the MOIs indicated in the figures. Media were replaced by freshmedia, and virus titers in the harvested samples were determined byplaque assay on BHK-21 cells.

Example 7 Analysis of Protein Synthesis

BHK-21 cells were electroporated with 4 mg of the indicated RNAs, andone-fifth of the electroporated cells were seeded into six-well Costarplates. At 12 h post transfection, proteins were metabolically labeledby incubating for 30 min in 0.8 ml of DMEM medium lacking methionine,supplemented with 0.1% FBS and 20 mCi/ml of [³⁵S]methionine. After thisincubation, they were scraped into the media, pelleted by centrifugationand dissolved in 100 μl of standard protein loading buffer. Equalamounts of proteins were loaded onto sodium dodecyl sulfate (SDS)-10%polyacrylamide gels. After electrophoresis, gels were dried andautoradiographed.

Example 8 RNA Analysis

To analyze the synthesis of virus-specific RNAs, cells wereelectroporated with 4 mg of the in vitro-synthesized viral RNAs, andone-fifth of the cells were seeded into 35-mm dishes as. At 4.5 h posttransfection, medium in the wells was replaced by 1 ml of aMEMsupplemented with 10% FBS, ActD (1 μg/ml) and [³H]uridine (20 mCi/ml).After 4 h of incubation at 37° C., total cellular RNAs were isolated byTrizol (Invitrogen) according the Manufacturer's protocol, thendenatured with glyoxal in dimethyl sulfoxide and analyzed by agarose gelelectrophoresis using described conditions (7). Gels were impregnatedwith 2,5-diphenyloxazole (PPO), dried and autoradiographed.

Example 9 IFN-a/b Assay

The concentrations of IFN-a/b in the media were measured by a describedbiological assay (41). Briefly, L929 cells were seeded in 100 ml ofcomplete media at a concentration of 5×10⁴ cells/well in 96-well platesand incubated at 37° C. for 6 h. Samples of media harvested frominfected NIH 313 cells were treated with UV light for 1 h, and seriallydiluted in two-fold steps directly in the wells with L929 cells. Afterincubation for 24 h at 37° C., an additional 100 ml of media with 2×10⁵PFU of vesicular stomatitis virus (VSV) was added to the wells andincubation continued for 36-40 h. Then cells were stained with crystalviolet, and the end point was determined as the concentration of IFNa/brequired to protect 50% of the cells from the VSV-induced CPE. TheIFN-a/b standard for normalization of the results was purchased from theATCC, and titers of the released viruses were determined by plaque assayon BHK-21 cells.

Example 10 Evaluation of Virus Replication in Mosquitoes

To assess replication competence in mosquitoes in vivo, intrathoracicinoculations of Aedes aegypti (a colony originating in Galveston, Tex.)mosquitoes using ca. 10⁵ PFU in a 1 μL volume were used. Intrathoracicinoculation was selected over oral exposure because nearly any culicinemosquito is highly susceptible to intrathoracic infection by anyalphavirus, while oral susceptibility is highly variable and much lesssensitive (42). Following inoculation using a glass pipette, mosquitoeswere incubated 10 days at 27° C. and then titrated individually in 1 mLof MEM supplemented with 20% FBS and Fungizone. A 100 μL volume of eachtitrated mosquito was then added to a Vero cell monolayer on a 24 wellplate and observed for 5 days for cytopathic effects to detectinfection. Assay controls included both the TC-83 parent virus and theIRES mutant.

Example 11 Immunization and Challenge with Virulent VEEV

Six-day-old NIH Swiss mice were inoculated intracerebrally (i.c.) withVEEV TC-83 strain or the designed mutants at a dose of ca. 10⁶ PFU in atotal volume of 20 μl of PBS. After infection, each cohort of 8-10animals was maintained for 2 months without any manipulation. For 21days, mice were observed daily for signs of illness (ruffled fur,depression, anorexia and/or paralysis) and/or death.

Eight-week-old female NIH Swiss mice were vaccinated s.c. at a dose ofca. 10⁶ PFU/mouse using VEEV TC-83 or the recombinant virus, thenchallenged subcutaneously 4 weeks later with ca. 10⁴ PFU of highlyvirulent VEEV strain 3908. For 21 days, mice were observed twice dailyfor signs of illness (ruffled fur, depression, anorexia and/orparalysis) and/or death.

Example 12 Recombinant VEEV TC-83-Based Viruses

The present invention developed alphaviruses capable of efficientreplication in the cells of vertebrate, but not of mosquito origin.Therefore, replication of such viruses had to depend on proteins or RNAsequences that function only in vertebrate, but not in insect cells. Toachieve this, the present invention designed a method to make expressionof alphavirus structural proteins dependent on the EMCV IRES. Thedesigned IRES did not contain the poly(C) sequence but retained thefirst 4 codons of EMCV polyprotein to achieve the most efficienttranslation of VEEV TC-83 structural genes. In later experiments, it wasconfirmed that these additional amino acids had no negative effect onvirus replication, but had detectable positive effect on the translationof viral structural proteins.

In one of the constructs, VEEV/IRES, the IRES sequence was cloned intothe subgenomic RNA downstream of the intact 5′UTR (FIG. 1A). Therefore,this genome was expected to be capable of subgenomic RNA synthesis. Inanother recombinant, VEEV/mutSG/IRES, the subgenomic promoter wasinactivated by 13 synonymous point mutations (FIGS. 1A and 1B), whichwere expected to prevent reversion to an active SG RNA promoter. Topromote synthesis of the VEEV structural proteins, the IRES sequence wascloned to replace the 26S 5′UTR.

The genome RNAs of VEEV/IRES, VEEV/mutSG/IRES and unmodified, wt VEEVTC-83 were synthesized in vitro and transfected into BHK-21 cells. Inthe infectious center assay, the VEEV/IRES RNA demonstrated the sameinfectivity as the RNA of TC-83, and developed plaques of a uniform sizesimilar to those of the TC-83 (FIGS. 1A and 1C). This was a strongindication that no additional, adaptive mutations were required forproductive replication of the designed virus. VEEV/IRES replicated totiters exceeding 10⁹ PFU/ml, but these final titers and virusreplication rates were significantly slower (FIG. 1D) than those of VEEVTC-83. BHK-21 cells transfected with another recombinant viral genome,VEEV/mutSG/IRES, which lacked the subgenomic promoter, producedinfectious virus very inefficiently (FIG. 1D). In the infectious centerassay, this construct developed mainly pinpoint plaques, and theirnumber was difficult to estimate. Surprisingly, this virus demonstratedfurther evolution upon serial passage and rapidly developed variantsthat produced larger plaques (FIG. 1C and data not shown). The growthcurve presented in FIG. 1D represents the release of both small andlarge plaque-forming viruses.

Thus, the results of these experiments indicated that, at least in thecontext of the VEEV/IRES genome, EMCV IRES could produce structuralproteins at levels sufficient for VEEV replication. The construct with amutated subgenomic promoter, VEEV/mutSG/IRES, produced adefective-in-replication virus that could evolve for more efficientreplication.

Example 13 Analysis of Adaptive Mutations in VEEV/mutSG/IRES

The evolution of VEEV/mutSG/IRES to a large plaque phenotype suggestedan accumulation of additional mutations in the viral genome. Thereversion to the wt genome sequence was an impossible event due to thelarge number of introduced point mutations, so the location of adaptivemutations was difficult to predict. To identify the mutations, 5 plaquesof VEEV/mutSG/IRES samples harvested at 24 h post electroporation wererandomly selected, and the entire genomes (including the 3′ and 5′UTRs)of two plaque-purified variants were sequenced. The list of themutations identified is presented in FIG. 2A. The majority of them weresynonymous and were not present in the known cis-acting RNA elements.Thus, their effect on virus replication was very unlikely. However, thegenomes of both plaque isolates contained the same mutation in the nsP2protein, Y₃₇₀→C, and one of the genomes had the next encoded aa changedas well (K₃₇₁→Q).

To test the effect of the mutations on virus replication, Y₃₇₀→C andboth Y₃₇₀→C and K₃₇₁→Q were cloned into the original VEEV/mutSG/IRESconstruct (FIG. 2B) and compared the RNA infectivity, virus replicationrates and plaque sizes with those of the original VEEV/mutSG/IRES andother constructs. The same mutations were also cloned into the VEEVTC-83 genome to test their effect on the replication of this parentalvirus. The IRES-encoding genome RNAs with either one or both mutationsin the genome, VEEV/mutSG/IRES/1 and VEEV/mutSG/IRES/2, demonstrated thesame infectivity in the infectious center assay as did VEEV TC-83 RNAand rescued viruses formed uniform plaque sizes similar to those of theVEEV TC-83. They also demonstrated a strong increase in the growth rates(FIGS. 2C and 1D), but the effect of the second mutation was barelydetectable. Thus, taken together, the data indicated that the Y₃₇₀→Cmutation in the nsP2 had a strong positive effect on virus replication,and the second mutation did not noticeably improve it. When introducedinto the VEEV TC-83 genome, the same mutations did not have anydetectable effect on the rates of virus replication or on final titers(FIG. 2C), suggesting that the replication enhancement was specific tothe VEEV/mutSG/IRES variant.

The identified aa changes (Y₃₇₀→C and K₃₇₁→Q) could represent only afraction of possible mutations leading to the efficient replication ofthe subgenomic promoter-deficient, IRES-containing virus. Therefore, inparallel experiments, nt 2161-2959 in the other three plaque clonesisolated from the samples were sequenced, harvested at 24 h post RNAtransfection of the in vitro-synthesized RNAs, and of 5 plaque-purifiedvariants isolated from the virus stock after an additional 3 passages inVero cells. It was anticipated that such passaging would lead to theselection of the most efficiently replicating viruses. The list of theidentified mutations is presented in FIG. 3A. Sequencing was performeddirectly from the RT-PCR-derived DNA fragments; therefore, the presentedmutations represent the consensus sequences in the plaque-derived viruspopulation and are not PCR-derived (FIG. 3B).

All of the isolates contained mutations in the sequenced fragment,corresponding to the carboxy terminal fragment of the RNA helicasedomain of nsP2, and all of the altered amino acids were located betweenamino acids 348-424. Moreover, the most common mutation, both in theoriginal viral stock, generated after electroporation, and in thepassaged pool, was Y₃₇₀→C. This was an indication that it likely has oneof the most prominent effects on replication; therefore, theabove-described variant with this particular mutation,VEEV/mutSG/IRES/1, was used in the experiments outlined in the followingsections.

Example 14 Effect of the nsP2 Y₃₇₀→C Mutation on Virus Replication

Identification of the adaptive mutation in the carboxy terminal fragmentof the nsP2-associated RNA helicase was surprising and did not suggestany obvious explanation for the increase in VEEV/mutSG/IRES replication.This mutation could possibly have a stimulatory effect either on RNAreplication, or the viral structural proteins translation, or viralparticle formation, or replicative complexes compartmentalization, etc.However, the most expected effect was an increase in viral RNAsynthesis. Therefore, BHK-21 cells were transfected with invitro-synthesized genomes of different VEEV variants, metabolicallylabeled the newly synthesized viral RNAs with [³H]uridine in thepresence of ActD for 4 h beginning 4.5 h post electroporation, and theRNA was analyzed by electrophoresis in agarose gels (FIG. 4A).

As expected, VEEV/IRES was capable of subgenomic RNA synthesis, whichindicated that the IRES introduced at the 3′ end of the subgenomic RNA5′UTR did not interfere with the subgenomic promoter activity.VEEV/mutSG/IRES and its variants with adaptive mutations in nsP2produced no detectable SG RNAs. Thus, 13 mutations introduced into thepromoter sequence of these genomes completely abolished thetranscription of the subgenomic RNA. Surprisingly, the adaptivemutations in the nsP2 did not have a noticeable effect on RNA genomereplication, and VEEV/mutSG/IRES/1 and VEEV/mutSG/IRES/2 genome RNAsreplicated as efficiently as did the originally designed VEEV/mutSG/IRESgenome. Moreover, the genome RNA replication of all of the variants wasvery similar to that of VEEV TC-83. No effect of these mutations wasdetected in the context of the original VEEV TC-83 RNA as well (seelanes corresponding to VEEV/1 and VEEV/2). This finding stronglysuggested that adaptation did not result in an increase in RNAreplication.

Synthesis of viral structural proteins was evaluated at 12 h postelectroporation (FIG. 4B). By that time, VEEV/IRES- andVEEV/mutSG/IRES-specific capsid and likely other structural proteinswere synthesized ˜2-fold less efficiently than in the cells transfectedwith VEEV TC-83 RNA. This reasonably small difference does not explainthe more than 4 and 7 orders of magnitude lower infectious titers ofVEEV/IRES and VEEV/mutSG/IRES viruses, respectively (compared to thetiters of VEEV TC-83), detected in samples harvested at 12 h posttransfection. Moreover, no difference between the synthesis of viralproteins in BHK-21 cells containing the original VEEV/mutSG/IRES genomesversus VEEV/mutSG/IRES/1 and VEEV/mutSG/IRES/2 with adaptive mutationsin the nsP2 was detected in this and other experiments. Thedistinguishing feature of the patterns of the labeled proteins in thecells infected with the IRES-containing viruses was in the presence oftwo additional bands, which were identified by mass spectrometry asheat-shock proteins Hsp90 and Hsp72. The biological significance oftheir induction is not clear yet, but might result from someabnormalities in viral structural protein(s) folding leading to stressdevelopment in the cells with the structural proteins expressed from theIRES.

In additional experiments, the intracellular distribution of the viralglycoproteins in cells infected with VEEV TC-83, VEEV/mutSG/IRES andVEEV/mutSG/IRES/1 were assessed and the presence of these proteins onthe cell surface was analyzed by staining with VEEV-specific antibodies.No noticeable difference in the distribution of the glycoproteins wasidentified. The possibility that the adaptive mutations caused theformation of an additional packaging signal in the viral genome wasexamined; the mutation-containing fragment (corresponding to nt2533-2950 of the VEEV genome) was cloned into the 3′ UTR ofVEEV/mutSG/IRES genome, and the recombinant in vitro-synthesized RNA wastested in the infectious center assay. No increase in plaque size orvirus titers, compared to those of the original VEEV/mutSG/IRES, wasdetected.

In another variant, a subgenomic promoter and a VEEV capsid-codingsequence was cloned into the 3′UTR of VEEV/mutSG/IRES genome to testwhether the additional capsid expression from the subgenomic RNA wouldincrease the efficiency of virus replication. This modification also didnot have any positive effect on virus titers. Last, whetherVEEV/mutSG/IRES produced genome-free subviral particles instead ofinfectious virus was analyzed. It was observed that this was not thecase: cells transfected with VEEV/mutSG/IRES RNA and metabolicallylabeled with [³⁵S]methionine did not produce subviral particles thatcould be detected by ultracentrifugation in sucrose gradients (data notshown). Thus, taken together, the above-described complex analysis didnot point to obvious mechanistic explanations for the very inefficientreplication of the original VEEV/mutSG/IRES or for the positive effectof the detected mutations in VEEV nsP2 on the replication of theIRES-containing virus. However, an aim of the present invention was thedevelopment of the VEEV variants, whose replication depends on the EMCVIRES function, and both VEEV/IRES and VEEV/mutSG/IRES/1 meets this goal.

Example 15 Replication of the IRES-Dependent VEEV Variants in theMosquito Cells and Mosquitoes

The accumulated data about alphavirus replication unambiguouslydemonstrate their genetic instability and high rate of evolution,resulting in the deletion of any heterologous genes (17, 40),particularly if they have a negative effect on virus replication.Therefore, whether the designed EMCV IRES insertions would be stable andrender the viruses incapable of replication in mosquito cells wasexamined. To test this, C₇10 mosquito cells were infected with VEEV/IRESand VEEV/mutSG/IRES viruses harvested at 24 h postelectroporation of thein vitro-synthesized RNAs into BHK-21 cells. VEEV/mutSG/IRES was usedinstead of the above-described VEEV/mutSG/IRES/1, with an adaptivemutation Y₃₇₀→C in the nsP2, to test the entire library of the variants,released after the RNA transfection, for the ability to establishreplication in mosquito cells.

On the first passage, at 48 h post infection of C₇10 cells the titer ofVEEV/IRES approached 1.5×10¹⁰ PFU/ml, and a similar titer was detectedin the stock, harvested after the second passage (FIGS. 5A and 5B). Thetiters of VEEV/mutSG/IRES, in contrast, were 150 PFU/ml after the firstpassage (this likely reflected residual virus used for infection ratherthan nascent virus produced in the mosquito cells), and below thedetection limit after the following second passage (FIGS. 5A and 5B). Inadditional experiments, the plaque-purified variants of VEEV/mutSG/IRESthat contained adaptive mutations in nsP2 were passaged in C₇10 cells.No infectious virus was ever recovered after two blind passages.

In another experiment, Ae. aegypti mosquitoes were intrathoracicallyinoculated with ca. 10⁵ PFU of VEEV TC-83 and the VEEV/mutSG/IRES/1variant. None of the 17 mosquitoes inoculated with the IRES mutantreplicated detectably in Ae. aegypti, whereas 17/17 mosquitoesinoculated with the TC-83 parent strain replicated to detectable levelsin the CFE assay, with a mean titer of over 10⁶ PFU/mosquito. Thus, theIRES-containing VEEV variant VEEV/mutSG/IRES/1 was incapable ofreplicating in mosquito cells both in vitro and in vivo.

To explain the high titers of VEEV/IRES (capable of producing thesubgenomic RNA variant) after passaging in mosquito cells, twoindividual plaques were randomly selected and the IRES-containingfragment of the genome was sequenced. In both isolates, the IRESsequence was no longer present in the viral genomes, as, and only 13(SEQ ID NO: 4) and 15 (SEQ ID NO: 5) residual nucleotides of theoriginal IRES were found (FIG. 5C). Thus, passaging of VEEV/IRES variantin mosquito cells led to an accumulation of the IRES-negative variants,and VEEV/mutSG/IRES (that lacked the subgenomic promoter) did notdevelop mutants capable of replicating efficiently in mosquito cells.

Example 16 VEEV/mutSG/IRES/1 Variant Demonstrates an AttenuatedPhenotype

The present invention was aimed at development of VEEV variantsincapable of replicating in cells of mosquito origin (and,correspondingly, in mosquito vectors) but demonstrating a moreattenuated phenotype in vertebrates than the parental VEEV TC-83. Theslower replication rates of VEEV/mutSG/IRES/1 variant raised a concernthat this virus might be incapable of replicating in vertebrate cellswith intact IFN-a/b production and signaling. However, this was not thecase. FIG. 6 demonstrates that VEEV/mutSG/IRES/1 replicated in the NIH3T3 cells, which have no defects in IFN-alpha/beta secretion andsignaling, to the titers above 10⁹ PFU/ml. Its replication caused a moreefficient IFN-a/b induction (FIG. 6), but apparently the IFN release didnot abrogate the already established virus replication. As shown inBHK-21 cells (FIG. 2C), replication of VEEV/mutSG/IRES/1 was lessefficient than that of the VEEV TC-83, suggesting that theIRES-dependent mutant might be attenuated in vivo. Indeed, after thei.c. inoculation of 6-day-old mice with ca. 10⁶ PFU. 86% survived theinfection and did not develop signs of encephalitis; in contrast, 92% ofmice were killed by the same dose of VEEV strain TC-83. Taken together,these data indicate that genetically modified, IRES-dependent VEEV wasmore attenuated than the parental VEEV TC-83.

Nevertheless, the VEEV/mutSG/IRES/1 variant remained immunogenic in bothneonatal and adult mice. Of the twelve 6-day-old mice that survived i.c.inoculation with VEEV/mutSG/IRES/1, 10 survived s.c. challenge with 10⁴PFU of wild-type VEEV strain 3908 administered 5 weeks later; incontrast, of 12 sham (PBS)-infected mice challenged in the same manner,none survived (FIG. 7). The VEEV/mutSG/IRES/1 was also immunogenic inadult mice; one s.c. immunization with ca. 10⁶ PFU protected 80% of miceagainst s.c. challenge 3 weeks later with 10⁴ PFU (˜10⁴ LD₅₀) of VEEVstrain 3908 (FIG. 8). Neutralizing antibody titers (PRNT₈₀) wereundetectable <1:20 in all of these mice immediately before challenge,suggesting that the incomplete protection after 1 vaccination was likelya result of lower level of IRES-containing virus replication in vivo.Thus, its high level of attenuation confers a high degree of safety, butrepeated vaccinations will be likely required.

Example 17 Expression Strategy to Reduce Attenuation but Maintain Lackof Mosquito Infectivity

In a separate embodiment of the present invention, a novel expressionstrategy was designed to reduce attenuation but maintain lack ofmosquito infectivity. This strategy involved placing the IRES downstreamof the envelope glycoprotein genes, with the capsid gene at the 3′ endof the subgenomic region just upstream of the 3′ UTR. (FIG. 9). Thus, asubgenomic message was made, with the envelope protein genes translatedin a cap-dependent manner but the capsid protein translated in anIRES-dependent manner. Replication of this new IRES mutant in BHK andVero cells was again efficient, but could not be detected in C7/10mosquito cells. Intrathoracic inoculation of 20 Aedes aegypti adultfemale mosquitoes yielded no evidence of replication. When this IRESversion 2 was used to vaccinate mice, all 10 seroconverted with meantiters about 2-fold lower than induced by normal TC-83, and all 10 micewere protected by IRES version 2 from lethal, subcutaneous challenge.Therefore, the new IRES expression strategy appears to result in lessattenuation while retaining the mosquito-incompetent phenotype.

TABLE 1 Immunogenicity and efficacy of TC-83 IRES mutants Fraction Meanprotected neutralizing against lethal Vaccine Fraction Ab titer ± VEEVstrain seroconverted SD challenge IRES  0/10 <20  8/10 version 1 IRES10/10 224 ± 260 10/10 version 2 TC-83 5/5 576 ± 143 5/5 Sham 0/5 <20 0/5

Example 18 Recombinant Chikungunya Based Viruses

Based on the attenuation strategy described supra, two (2) chikungunyavirus mutants were designed, that employed the encephalomyocarditisvirus (EMCV) internal robosome entry site to alter gene expression. Thefirst version CHIKV/IRESv1 (FIG. 10) contained 13 synonymous mutationsto eliminate activity of the subgenomic promoter, and the addition ofthe IRES in the intergenic region upstream of the structural proteinopen reading frame (ORF). The second version CHIKV/IRESv2 (FIG. 10)retained the subgenomic promoter but positioned the capsid protein genedownstream of the envelope protein genes and behind the IRES.

Viruses rescued from these constructs were tested for attenuation in thebaby CD1 mouse model (46) and compared to the parent La Reunion (LR)CHIKV strain as well as a vaccine strain developed by the U.S. Army (45)181/25 that was highly immunogenic in human volunteers but showed somereactivity (44). The results demonstrated that both IRES mutantsproduced less viremia (FIG. 11) and less replication in the legs (FIG.12) or brains (FIG. 13) of 6-day-old mice compared for the LR or 181/25strains.

Example 19 Vaccinated with CHIKV/IRESv1 and CHIKV/IRESv2

Adult CD1 mice were vaccinated with the CHIKV/IRES versions 1 or 2described supra, and compared with 181/25 for their ability to protectfrom intranasal challenge 4 weeks later with the Ross CHIKV strain. All3 vaccine strains provided complete protection, which was significantlybetter than sham vaccination (FIG. 14). Neutralizing antibody titerswere similar for cohorts immunized with CHIK/IRESv1 and v2, as well as181/25 (Table 2). These results indicate that the IRES attenuationapproach for CHIK produces better attenuation and equivalentimmunogenicity and protection compared to the U.S. Army 181/25 strain.

TABLE 2 Seroconversion of Outbred Mice After Vaccination PRNT₈₀ titerMouse Number CHIKV-IRESv1 CHIKV-IRESv2 181/25 1 80 40 80 2 40 80 80 3 4080 80 4 80 160 80 5 40 40 40 6 160 80 80 7 20 80 40 8 20 40 40 9 80 8080 10  80 % Seroconversion 100 100 100 Mean titer 62 76 67 STD 44 35 20Vaccination with 10⁵ PFU, single dose

Example 20 Evaluation of the Ability of CHIK/IRESv1 Virus to InfectC6/36 Mosquito Cells

CHIK/IRESv1 was also tested for its ability to infect C6/36 mosquitocells or Ae. aegypti mosquitoes to determine environmental safety. After5 blind, serial passages in mosquito cells with inoculum titers of ca.105 Vero cell plaque forming units, no evidence of viral replication wasdetected by cytopathic effect assays or RT-PCR; A positive control ofthe LR CHIKV strain replicated as expected. Cohorts of 20 Ae. aegyptiwere also inoculated intrathoracically with ca 104 Vero cell plaqueforming units, no infection was detected by cytopathic effect assays orRT-PCR for CHIK/IRESv1, while the LR strain replicated in all 20inoculated mosquitoes. These results indicate that the CHIK/IRESv1 isincapable of replicating in mosquitoes or mosquito cells.

The following references were cited herein:

-   1. Aguilar, P. V. et al., 2007, J Virol 81:3866-76.-   2. Alevizatos, A. C. et al., 1967, Am J Trop Med Hyg 16:762-8.-   3. Barton, D. J. et al., 1999, J Virol 73:10104-12.-   4. Berge, T. O. et al., 1961, Am. J. Hyg. 73:209-218.-   5. Blaney, J. E., Jr. et al., 2006, Viral Immunol 19:10-32.-   6. Blaney, J. E., Jr. et al., 2005, J Virol 79:5516-28.-   7. Bredenbeek, P. J. et al., 1993, J. Virol. 67:6439-6446.-   8. Burke, D. S. et al., 1977, J Infect Dis 136:354-9.-   9. Dal Canto, M. C., and S. G. Rabinowitz, 1981, J Neurol Sci    49:397-418.-   10. Davis, N. L. et al., 1995, Virology 212:102-110.-   11. Davis, N. L. et al. 1991, Virology 183:20-31.-   12. Davis, N. L. et al., 1989, Virology 171:189-204.-   13. Garmashova, N. et al., 2007. J Virol 81:13552-65-   14. Garmashova, N. et al. 2006, J Virol 80:5686-96.-   15. Garmashova, N. et al., 2007, J Virol 81:2472-84.-   16. Gorchakov, R. et al., 2004, J Virol 78:61-75.-   17. Gorchakov, R. et al., 2007, Virology 366:212-25.-   18. Griffin, D. 2001. Alphaviruses, p. 917-962. In Knipe and Howley    (ed.), Fields' Virology, Fourth Edition. Lippincott, Williams and    Wilkins, NY.-   19. Hart, M. K. et al., 2000, Vaccine 18:3067-75.-   20. Jan, E., and P. Sarnow. 2002, J Mol Biol 324:889-902.-   21. Johnson, K. and D. Martin. 1974. Adv. Vet. Sci. Comp. Med.    18:79-116.-   22. Khromykh, A. A. et al., 2001, J Virol 75:4633-40.-   23. Kinney, R. M. et al. 1993, J. Virol. 67:1269-1277.-   24. Kinney, R. M. et al., 1989, Virology 170:19-30.-   25. Kuhn, R. J. et al., 1996, J Virol 70:7900-9.-   26. Lemm, J. A. et al., 1990, J. Virol. 64:3001-3011.-   27. Liljeström, P. et al., 1991, J. Virol. 65:4107-4113.-   28. Morrill, J. C. et al., 1991, Vaccine 9:35-41.-   29. Murphy, et al. 2001. Immunization against viral diseases, p.    435-467. In D. Knipe and Howley (ed.), Fields' Virology, 4th Ed.    Lippincott, Williams and Wilkins, New York.-   30. Nugent, C. I. et al., 1999, J Virol 73:427-35.-   31. Paessler, S. et al., 2003, J Virol 77:9278-86.-   32. Pedersen, C. E. et al., 1972, Am J Epidemiol 95:490-6.-   33. Petrakova, O. et al., 2005, J Virol 79:7597-608.-   34. Pittman, P. R. et al., 1996, Vaccine 14:337-43.-   35. Pugachev, K. V. et al., 2000, J Virol 74:10811-5.-   36. Rice, C. M. et al., 1987, J. Virol. 61:3809-3819.-   37. Rivas, F. et al., 1997, J Infect Dis 175:828-32.-   38. Russo, A. T. et al., 2006, Structure 14:1449-58.-   39. Strauss, J. H., and E. G. Strauss, 1994, Microbiol. Rev.    58:491-562.-   40. Thomas, J. M. et al., 2003, J Virol 77:5598-606.-   41. Trgovcich, J. et al., 1996. Virology 224:73-83.-   42. Weaver, S. C. 1997. Vector Biology in Viral Pathogenesis, p.    329-352. In N. Nathanson (ed.), Viral Pathogenesis.    Lippincott-Raven, New York.-   43. White, L. J. et al., 2001, J Virol 75:3706-18.-   44. Edelman, R. et al. (2000). Am J Trop Med Hyg 62(6), 681-5.-   45. Levitt, N. H. et al. (1986). Vaccine 4(3), 157-62.-   46. Ziegler, S. A. et al. (2008). Am J Trop Med Hyg 79(1), 133-9.

Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. Further, these patents and publications areincorporated by reference herein to the same extent as if eachindividual publication was specifically and individually indicated to beincorporated by reference.

1-36. (canceled)
 37. An attenuated, recombinant alphavirus comprising:a) a capsid gene positioned downstream from the envelope glycoproteingenes and upstream from the 3′ UTR of a subgenomic RNA in the genome ofthe alphavirus; and b) an internal ribosomal entry site (IRES)introduced between the end of the envelope glycoprotein genes and thepositioned capsid gene, wherein the capsid gene is expressed in acap-independent manner, wherein the capsid protein is translated in anIRES-dependent manner, wherein the envelope glycoprotein genes aretranslated in a cap-dependent manner, and wherein the alphavirus isattenuated.
 38. The attenuated, recombinant alphavirus of claim 37,wherein the alphavirus is incapable of replicating in mosquitoes andmosquito cells.
 39. The attenuated, recombinant alphavirus of claim 37,wherein the alphavirus is Chikungunya virus, Eastern Equine EncephalitisVirus, Venezuelan Equine Encephalitis virus, or Western EquineEncephalitis virus.
 40. The attenuated, recombinant alphavirus of claim37, wherein the attenuated, recombinant alphavirus further comprises aninactivated subgenomic promoter.
 41. An expression vector comprising anucleotide sequence encoding the attenuated, recombinant alphavirus ofclaim
 37. 42. An isolated host cell comprising the expression vector ofclaim
 41. 43. A pharmaceutical composition comprising the attenuated,recombinant alphavirus of claim 37 and a pharmaceutically acceptablecarrier.
 44. A method for inducing an anti-alphavirus immune response ina subject, comprising administering to the subject a composition ofclaim
 43. 45. The method of claim 44, wherein the subject is a human, ahorse or other domestic animal.
 46. A method for making an alphavirusthat is incapable of replicating in mosquitoes and mosquito cells, themethod comprising: introducing a mutation into an alphavirus genome, themutation inactivating cap-dependent translation of a structural proteinof the alphavirus; and cloning an internal ribosomal entry site thatselectively initiates translation in cells of vertebrate origin into thealphavirus genome upstream of a gene that encodes the structuralprotein.
 47. The method of claim 46, wherein the step of introducing amutation includes inactivating a promoter necessary for replication ofthe alphavirus in a mosquito.
 48. The method of claim 46, wherein thestructural protein is the capsid protein of the alphavirus and the stepof introducing a mutation includes repositioning a gene encoding thecapsid protein downstream of a gene encoding an envelope glycoprotein ofthe alphavirus.
 49. The method of claim 48, wherein the internalribosomal entry site is positioned between a gene encoding an envelopeprotein and the gene encoding the capsid protein.
 50. An attenuated,recombinant alphavirus comprising: a) an alphavirus genome having amutation that inactivates cap-dependent translation of a structuralprotein of the alphavirus; and b) an internal ribosomal entry site(IRES) that selectively initiates translation in cells of vertebrateorigin positioned upstream of a gene that encodes the structuralprotein, and wherein the alphavirus is attenuated.
 51. The attenuated,recombinant alphavirus of claim 50, further comprising an inactivatedsubgenomic promoter.
 52. The attenuated, recombinant alphavirus of claim51, wherein the structural protein is the capsid protein of thealphavirus and the mutation includes a gene encoding the capsid proteinpositioned downstream of a gene encoding an envelope glycoprotein of thealphavirus.
 53. The attenuated, recombinant alphavirus of claim 55,wherein the IRES is positioned between a gene encoding an envelopeprotein and the gene encoding the capsid protein.
 54. A pharmaceuticalcomposition comprising the attenuated, recombinant alphavirus of claim50 and a pharmaceutically acceptable carrier.
 55. A method for inducingan anti-alphavirus immune response in a subject, comprisingadministering to the subject the composition of claim
 54. 56. The methodof claim 54, wherein the subject is a human, a horse or other domesticanimal.